Abstract: 【Objective】To investigate the difference of gene expression in Raphanus sativus seedlings responding to the high temperature through transcriptome sequencing analysis, so as to provide theoretical basis for the molecular mechanism of tolerance to heat stress in R. sativus.【Method】 The heat-resistant radish variety 1116T and heat-sensitive radish variety Wr129 were treated with high temperature(40℃)stress for 0(room temperature,control)and 24 h. The total RNA of samples at each time point was extracted for transcriptome sequencing. Taking|log₂ Fold Change| ≥ 1,P<0.05 and TPM ≥ 10 as the standard,the differentially expressed genes(DEGs)were screened,and GO function and KEGG metabolic pathway enrichment analysis were performed.【Result】 With the increase of heat stress time, the stems and leaves of Wr129 and 1116T seedlings gradually wilted. After 24 h of stress, the leaves of Wr129 wilted,drooped and became dry and yellow, while the leaves of 1116T were slightly wilted, drooped and remained green. A total of 81.45 Gb clean data were obtained from 12 libraries, and on average of 62.30% of the reads were mapped to the reference genome. In total, 2701 DEGs were screened. Through GO functional annotation analysis, 2629 DEGs were annotated in the GO database, of which 559 DEGs were enriched to three items of chloroplast envelope, chloroplast stroma and chloroplast thylakoid in cellular component categories, and 470 DEGs were annotated as related to stress response in biological process. KEGG metabolic pathway enrichment showed that 1166 DEGs were annotated into 124 metabolic pathways, of which 74 DEGs were enriched in photosynthetic pathway and the photosynthesis-antenna protein pathway, 23 DEGs were enriched in the porphyrin and chlorophyll metabolic pathway, and most genes were induced under heat stress. While 35 DEGs involved in the glutathione metabolism pathway, and 14 glutathione-S-transferase genes(GSTs)up-regulated in this pathway. The maximum photochemical efficiency(F_v/F_m)and total chlorophyll content of PSII in leaves of Wr129 and 1116T seedlings decreased under high temperature stress for 24 h,but the decrease of 1116T was small.【Conclusion】 The heat tolerance of Wr129 and 1116T is obviously different, because 1116T can improve its heat resistance by the regulation of metabolic process relating to antioxidant and photosynthetic systems and slow the F_v/F_m value and chlorophyll content rate.